| Epidemiological studies support that chronic periodontal infections are associated with increased risk of atherosclerosis. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic mice, while a major fimbria-deficient (FimA-) mutant did not. We also demonstrated that endothelial cells infected with fimbriated P. gingivalis exhibit increased cytokine/chemokine production and up-regulate expression of Toll-like receptors (TLRs). However, the mechanism(s) by which both P. gingivalis major and minor fimbria activate TLRs are poorly defined. In this study, we utilized 41-kDa (major) and 67-kDa (minor) fimbria mutants to demonstrate that major fimbria are required for P. gingivalis invasion of human aortic endothelial cells (HAEC). ELISA revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of the major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. The major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. We also demonstrated that HAEC express surface TLR4, while the majority of TLR2 is intracellular. Transient transfection of non-signaling forms of TLR2 or TLR4, or monoclonal antibody against TLR4 reduced IL-8 production by HAEC. We also demonstrated specific, saturable binding of major and minor fimbria to chimeric TLR2, but not TLR4 fusion proteins. Our results indicate that fimbria activate HAEC through direct interaction with TLR2 and indirectly via TLR4. Collectively, these data support that invasive P. gingivalis and fimbria stimulate TLR-mediated endothelial cell activation, a necessary initial event in the development of atherogenesis. |
